The present invention relates to ice-nucleators and, more particularly, to microbially produced ice-nucleators which are either whole cell or cell-free.
It has been known for years that certain microorganisms are capable of acting as nucleating agents for the formation of ice. A number of practical applications exist for exploiting such ice nucleating ability including inducing precipitation (e.g., cloud seeding) and snowmaking. Furthermore, the role of ice nucleating microorganisms in inducing frost injury to plants has been investigated. To date, the art has employed both the microorganisms themselves (whole cell products) and products derived from such microorganisms (cell free products).
When the microorganisms themselves are employed to form a whole cell product, it has been found desirable in cloud seeding to provide such microorganisms in a dried form because dried microorganisms can act as very efficient condensation nuclei which adsorb water very readily at low levels of water vapor supersaturation. Thus, U.S. Pat. No. 4,706,463 relates to the recovery of microorganisms having ice nucleating activity, in dried form. Prior methods of culturing microorganisms having ice nucleating activity were discussed. Such methods, while acknowledged to enable production of large volumes of microorganisms, were said to be inadequate in producing a dried product because much of the activity is lost during the drying of large volumes of the material. A process was therefore proposed for preserving the ice nucleating activity after drying of any suspension containing the microorganisms. Such method involves the steps of (a) bringing the temperature of said medium to a temperature of about 15.degree. C. or less, (b) forming a concentrate of the microorganism preferably having a water content of about 15-27%, while maintaining the temperature of about 15.degree. C. or less, (c) running the concentrate into a cryogenic liquid in the form of a fine stream so as to form frozen pellets of the concentrate preferably having a diameter of about 2-10 mm, and (d) freeze drying the pellets at a temperature below 25.degree. C.
Another whole cell product including ice nucleating activity is produced by fermenting a microorganism having ice nucleating activity. The microorganism is grown at a temperature of at least about 29.degree. C. in a medium until the stationary phase. Fermentation is continued during the stationary phase at a temperature below about 24.degree. C. The amount of nitrogen source in the growth medium should be low enough so that, at the conclusion of the growth phase, there is insufficient nitrogen source remaining to inhibit the formation of ice nucleating activity during the subsequent phase. It was found that INA is produced predominantly during the stationary phase of the fermentation if the temperature during such phase is maintained below 24.degree. C. Suitable microorganisms that have ice nucleation activity include Pseudomonas such as P. syringae and P. fluorescens, P. coronafaciens and P. pisi. Other microorganisms that are useful include Erwinia herbicola.
As previously indicated, methods have also been proposed for preparing cell free ice nucleating agents. For example, a cell-free method has been developed that helps to increase the ice nucleation active particle number per gram of a dried bacterial culture. The method entails the fluid energy mill grinding of a dry bacterial culture in order to produce a dry talcum like powder that approaches near single cell size distribution upon aerosolization. This is advantageous to the cloud seeding industry since it increases the number of active ice nuclei per particle of seeding material.
U.S. Pat. No. 4,464,473 describes isolation of DNA segments encoding for substances having ice nucleation activity (INA). The DNA is isolated from organisms known to provide for ice nucleation such as various species of Pseudomonas including syringe, coronafaciens, pisi, tabaci or fluorescens. Xanthomonas, such as translucens or Erwinia, such as herbicola can also be employed. The host is then transformed with the DNA and the substances having ice nucleation activity expressed by the host microorganism. According to the disclosure, organisms which have a wide variety of ecological niches can be modified so as to provide for ice nucleation activity in new environments and/or with higher efficiency.
Phelps, et al., in "Release of Cell-Free Ice Nuclei by Erwinia herbicola", J. Bact. 167(2): 496-502 (1986), employs Erwinia herbicola or Pseudomonas syringae for probing cell surfaces for ice nuclei. Cell free ice nuclei were isolated by growing the bacteria in DM glucose medium at 15.degree. C. to stationary phase and by collecting supernatant by centrifugation and filtration through a 0.22 m.mu. filter.
The advances in various whole cell and cell-free products notwithstanding, the art continues to seek out ice nucleators which exhibit a number of desirable properties. Included among such properties are (1) a high number of ice nucleating sites per gram of material; (2) a high number of ice nucleating sites per milliliter of material; (3) a high amount of ice nucleating activity per gram of protein; and (4) a high degree of stability at 37.degree. C.